Lung Development Genes and Adult Lung Function.

Rationale: Poor lung health in adult life may occur partly through suboptimal growth and development, as suggested by epidemiological evidence pointing to early life risk factors.Objectives: To systematically investigate the effects of lung development genes on adult lung function.Methods: Using UK Biobank data, we tested the association of 391 genes known to influence lung development with FVC and FEV1/FVC. We split the dataset into two random subsets of 207,616 and 138,411 individuals, using the larger subset to select the most promising signals and the smaller subset for replication.Measurements and Main Results: We identified 55 genes, of which 36 (16 for FVC, 19 for FEV1/FVC, and one for both) had not been identified in the largest, most recent genome-wide study of lung function. Most of these 36 signals were intronic variants; expression data from blood and lung tissue showed that the majority affect the expression of the genes they lie within. Further testing of 34 of these 36 signals in the CHARGE and SpiroMeta consortia showed that 16 replicated after Bonferroni correction and another 12 replicated at nominal significance level. Of the 55 genes, 53 fell into four biological categories whose function is to regulate organ size and cell integrity (growth factors; transcriptional regulators; cell-to-cell adhesion; extracellular matrix), suggesting that these specific processes are important for adult lung health.Conclusions: Our study demonstrates the importance of lung development genes in regulating adult lung function and influencing both restrictive and obstructive patterns. Further investigation of these developmental pathways could lead to druggable targets.

Mechanism of lung development in the aetiology of adult congenital pulmonary airway malformations.

Congenital pulmonary airway malformations (CPAMs) are rare lung abnormalities that result in cyst formation and are associated with respiratory distress in infants and malignant potential in adults. The pathogenesis of CPAMs remains unknown but data suggest disruption of the normal proximo-distal programme of airway branching and differentiation. Here, we demonstrate that adult human CPAM are lined with epithelium that retains SOX-2 and thyroid transcription factor-1 immunohistochemical markers, characteristic of the developing lung. However, RALDH-1, another key marker, is absent. This suggests a more complex aetiology for CPAM than complete focal arrest of lung development and may provide insight to the associated risk of malignancy.

Regenerative pharmacology for COPD: breathing new life into old lungs.

Chronic obstructive pulmonary disease (COPD) is a major global health concern with few effective treatments. Widespread destruction of alveolar tissue contributes to impaired gas exchange in severe COPD, and recent radiological evidence suggests that destruction of small airways is a major contributor to increased peripheral airway resistance in disease. This important finding might in part explain the failure of conventional anti-inflammatory treatments to restore lung function even in patients with mild disease. There is a clear need for alternative pharmacological strategies for patients with COPD/emphysema. Proposed regenerative strategies such as cell therapy and tissue engineering are hampered by poor availability of exogenous stem cells, discouraging trial results, and risks and cost associated with surgery. An alternative therapeutic approach is augmentation of lung regeneration and/or repair by biologically active factors, which have potential to be employed on a large scale. In favour of this strategy, the healthy adult lung is known to possess a remarkable endogenous regenerative capacity. Numerous preclinical studies have shown induction of regeneration in animal models of COPD/emphysema. Here, we argue that given the widespread and irreversible nature of COPD, serious consideration of regenerative pharmacology is necessary. However, for this approach to be feasible, a better understanding of the cell-specific molecular control of regeneration, the regenerative potential of the human lung and regenerative competencies of patients with COPD are required.

Platypnoea-orthodeoxia syndrome: beware of investigations undertaken supine.

Platypnoea-orthodeoxia syndrome (POS) is a rare disorder, manifesting as deoxygenation occurring when the patient is in the upright position. Four broad mechanisms for the condition have been described: intracardiac shunts, intrapulmonary shunts, hepatopulmonary syndrome and pulmonary ventilation-perfusion mismatch. Here, we present the first case of POS in a patient with a proven right to left intracardiac shunt occurring in the context of postural hypotension and normal right heart pressures. We highlight the need to carry out investigations in the upright position before discounting intracardiac shunting as a cause for the syndrome. Short-term improvement of the syndrome was obtained with medical management of the patient's orthostatic hypotension and as such suggests a conservative management strategy for similar patients, which may delay the need for invasive procedures to close the anatomical defect.

Novel anti-tumour necrosis factor receptor-1 (TNFR1) domain antibody prevents pulmonary inflammation in experimental acute lung injury.

BACKGROUND: Tumour necrosis factor alpha (TNF-α) is a pleiotropic cytokine with both injurious and protective functions, which are thought to diverge at the level of its two cell surface receptors, TNFR1 and TNFR2. In the setting of acute injury, selective inhibition of TNFR1 is predicted to attenuate the cell death and inflammation associated with TNF-α, while sparing or potentiating the protective effects of TNFR2 signalling. We developed a potent and selective antagonist of TNFR1 (GSK1995057) using a novel domain antibody (dAb) therapeutic and assessed its efficacy in vitro, in vivo and in a clinical trial involving healthy human subjects. METHODS: We investigated the in vitro effects of GSK1995057 on human pulmonary microvascular endothelial cells (HMVEC-L) and then assessed the effects of pretreatment with nebulised GSK1995057 in a non-human primate model of acute lung injury. We then tested translation to humans by investigating the effects of a single nebulised dose of GSK1995057 in healthy humans (n=37) in a randomised controlled clinical trial in which subjects were subsequently exposed to inhaled endotoxin. RESULTS: Selective inhibition of TNFR1 signalling potently inhibited cytokine and neutrophil adhesion molecule expression in activated HMVEC-L monolayers in vitro (P<0.01 and P<0.001, respectively), and also significantly attenuated inflammation and signs of lung injury in non-human primates (P<0.01 in all cases). In a randomised, placebo-controlled trial of nebulised GSK1995057 in 37 healthy humans challenged with a low dose of inhaled endotoxin, treatment with GSK1995057 attenuated pulmonary neutrophilia, inflammatory cytokine release (P<0.01 in all cases) and signs of endothelial injury (P<0.05) in bronchoalveolar lavage and serum samples. CONCLUSION: These data support the potential for pulmonary delivery of a selective TNFR1 dAb as a novel therapeutic approach for the prevention of acute respiratory distress syndrome. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT01587807.

AJRCCM: 100-Year Anniversary. Homeward Bound: A Centenary of Home Mechanical Ventilation.

The evolution of home mechanical ventilation is an intertwined chronicle of negative and positive pressure modes and their role in managing ventilatory failure in neuromuscular diseases and other chronic disorders. The uptake of noninvasive positive pressure ventilation has resulted in widespread growth in home ventilation internationally and fewer patients being ventilated invasively. As with many applications of domiciliary medical technology, home ventilatory support has either led or run in parallel with acute hospital applications and has been influenced by medical and societal shifts in the approach to chronic care, the creation of community support teams, a preference of recipients to be treated at home, and economic imperatives. This review summarizes the trends and growing evidence base for ventilatory support outside the hospital.

Deficient retinoid-driven angiogenesis may contribute to failure of adult human lung regeneration in emphysema.

BACKGROUND: Molecular pathways that regulate alveolar development and adult repair represent potential therapeutic targets for emphysema. Signalling via retinoic acid (RA), derived from vitamin A, is required for mammalian alveologenesis, and exogenous RA can induce alveolar regeneration in rodents. Little is known about RA signalling in the human lung and its potential role in lung disease. OBJECTIVES: To examine regulation of human alveolar epithelial and endothelial repair by RA, and characterise RA signalling in human emphysema. METHODS: The role of RA signalling in alveolar epithelial repair was investigated with a scratch assay using an alveolar cell line (A549) and primary human alveolar type 2 (AT2) cells from resected lung, and the role in angiogenesis using a tube formation assay with human lung microvascular endothelial cells (HLMVEC). Localisation of RA synthetic (RALDH-1) and degrading (cytochrome P450 subfamily 26 A1 (CYP26A1)) enzymes in human lung was determined by immunofluorescence. Regulation of RA pathway components was investigated in emphysematous and control human lung tissue by quantitative real-time PCR and Western analysis. RESULTS: RA stimulated HLMVEC angiogenesis in vitro; this was partially reproduced with a RAR-α agonist. RA induced mRNA expression of vascular endothelial growth factor A (VEGFA) and VEGFR2. RA did not modulate AT2 repair. CYP26A1 protein was identified in human lung microvasculature, whereas RALDH-1 partially co-localised with vimentin-positive fibroblasts. CYP26A1 mRNA and protein were increased in emphysema. CONCLUSIONS: RA regulates lung microvascular angiogenesis; the endothelium produces CYP26A1 which is increased in emphysema, possibly leading to reduced RA availability. These data highlight a role for RA in maintenance of the human pulmonary microvascular endothelium.

Choking on a foreign body: a physiological study of the effectiveness of abdominal thrust manoeuvres to increase thoracic pressure.

The Heimlich manoeuvre is a well-known intervention for the management of choking due to foreign body airway occlusion, but the evidence base for guidance on this topic is limited and guidelines differ. We measured pressures during abdominal thrusts in healthy volunteers. The angle at which thrusts were performed (upthrust vs circumferential) did not affect intrathoracic pressure. Self-administered abdominal thrusts produced similar pressures to those performed by another person. Chair thrusts, where the subject pushed their upper abdomen against a chair back, produced higher pressures than other manoeuvres. Both approaches should be included in basic life support teaching.

Endobronchial valves for patients with heterogeneous emphysema and without interlobar collateral ventilation: open label treatment following the BeLieVeR-HIFi study.

Outcomes in early trials of bronchoscopic lung volume reduction using endobronchial valves for the treatment of patients with advanced emphysema were inconsistent. However improvements in patient selection with focus on excluding those with interlobar collateral ventilation and homogeneous emphysema resulted in significant benefits in the BeLieVeR-HIFi study compared with sham treated controls. In this manuscript we present data from the control patients in the BeLieVeR-HIFi study who went on to have open label endobronchial valve treatment after completion of the clinical trial (n=12), combined with data from those in the treatment arm who did not have collateral ventilation (n=19). Three months after treatment FEV1 increased by 27.3 (36.4)%, residual volume reduced by 0.49 (0.76) L, the 6 min walk distance increased by 32.6 (68.7) m and the St George Respiratory Questionnaire for COPD score improved by 8.2 (20.2) points. These data extend the evidence for endobronchial valve placement in appropriately selected patients with COPD. TRIAL REGISTRATION NUMBER: ISRCTN04761234; Results.

Bronchoscopic lung volume reduction with endobronchial valves for patients with heterogeneous emphysema and intact interlobar fissures (the BeLieVeR-HIFi study): a randomised controlled trial.

BACKGROUND: Lung volume reduction surgery improves survival in selected patients with emphysema, and has generated interest in bronchoscopic approaches that might achieve the same effect with less morbidity and mortality. Previous trials with endobronchial valves have yielded modest group benefits because when collateral ventilation is present it prevents lobar atelectasis. METHODS: We did a single-centre, double-blind sham-controlled trial in patients with both heterogeneous emphysema and a target lobe with intact interlobar fissures on CT of the thorax. We enrolled stable outpatients with chronic obstructive pulmonary disease who had a forced expiratory volume in 1 s (FEV1) of less than 50% predicted, significant hyperinflation (total lung capacity >100% and residual volume >150%), a restricted exercise capacity (6 min walking distance <450 m), and substantial breathlessness (MRC dyspnoea score ≥3). Participants were randomised (1:1) by computer-generated sequence to receive either valves placed to achieve unilateral lobar occlusion (bronchoscopic lung volume reduction) or a bronchoscopy with sham valve placement (control). Patients and researchers were masked to treatment allocation. The study was powered to detect a 15% improvement in the primary endpoint, the FEV1 3 months after the procedure. Analysis was on an intention-to-treat basis. The trial is registered at controlled-trials.com, ISRCTN04761234. FINDINGS: 50 patients (62% male, FEV1 [% predicted] mean 31·7% [SD 10·2]) were enrolled to receive valves (n=25) or sham valve placement (control, n=25) between March 1, 2012, and Sept 30, 2013. In the bronchoscopic lung volume reduction group, FEV1 increased by a median 8·77% (IQR 2·27-35·85) versus 2·88% (0-8·51) in the control group (Mann-Whitney p=0·0326). There were two deaths in the bronchoscopic lung volume reduction group and one control patient was unable to attend for follow-up assessment because of a prolonged pneumothorax. INTERPRETATION: Unilateral lobar occlusion with endobronchial valves in patients with heterogeneous emphysema and intact interlobar fissures produces significant improvements in lung function. There is a risk of significant complications and further trials are needed that compare valve placement with lung volume reduction surgery. FUNDING: Efficacy and Mechanism Evaluation Programme, funded by the Medical Research Council (MRC) and managed by the National Institute for Health Research (NIHR) on behalf of the MRC-NIHR partnership.

A Novel Method for Floxed Gene Manipulation Using TAT-Cre Recombinase in Ex Vivo Precision-Cut Lung Slices (PCLS).

Precision-cut lung slices (PCLS), ex vivo 3D lung tissue models, have been widely used for various applications in lung research. PCLS serve as an excellent intermediary between in vitro and in vivo models because they retain all resident cell types within their natural niche while preserving the extracellular matrix environment. This protocol describes the TReATS (TAT-Cre recombinase-mediated floxed allele modification in tissue slices) method that enables rapid and efficient gene modification in PCLS derived from adult floxed animals. Here, we present detailed protocols for the TReATS method, consisting of two simple steps: PCLS generation and incubation in a TAT-Cre recombinase solution. Subsequent validation of gene modification involves live staining and imaging of PCLS, quantitative real-time PCR, and cell viability assessment. This four-day protocol eliminates the need for complex Cre-breeding, circumvents issues with premature lethality related to gene mutation, and significantly reduces the use of animals. The TReATS method offers a simple and reproducible solution for gene modification in complex ex vivo tissue-based models, accelerating the study of gene function, disease mechanisms, and the discovery of drug targets. Key features • Achieve permanent ex vivo gene modifications in complex tissue-based models within four days. • Highly adaptable gene modification method that can be applied to induce gene deletion or activation. • Allows simple Cre dosage testing in a controlled ex vivo setting with the advantage of using PCLS generated from the same animal as true controls. • With optimisation, this method can be applied to precision-cut tissue slices of other organs.

Deciphering the impacts of modulating the Wnt-planar cell polarity (PCP) pathway on alveolar repair.

Many adult lung diseases involve dysregulated lung repair. Deciphering the molecular and cellular mechanisms that govern intrinsic lung repair is essential to develop new treatments to repair/regenerate the lungs. Aberrant Wnt signalling is associated with lung diseases including emphysema, idiopathic pulmonary fibrosis and pulmonary arterial hypertension but how Wnt signalling contributes to these diseases is still unclear. There are several alternative pathways that can be stimulated upon Wnt ligand binding, one of these is the Planar Cell Polarity (PCP) pathway which induces actin cytoskeleton remodelling. Wnt5a is known to stimulate the PCP pathway and this ligand is of particular interest in regenerative lung biology because of its association with lung diseases and its role in the alveolar stem cell niche. To decipher the cellular mechanisms through which Wnt5a and the PCP pathway affect alveolar repair we utilised a 3-D ex-vivo model of lung injury and repair, the AIR model. Our results show that Wnt5a specifically enhances the alveolar epithelial progenitor cell population following injury and surprisingly, this function is attenuated but not abolished in Looptail (Lp) mouse lungs in which the PCP pathway is dysfunctional. However, Lp tracheal epithelial cells show reduced stiffness and Lp alveolar epithelial cells are less migratory than wildtype (WT), indicating that Lp lung epithelial cells have a reduced capacity for repair. These findings provide important mechanistic insight into how Wnt5a and the PCP pathway contribute to lung repair and indicate that these components of Wnt signalling may be viable targets for the development of pro-repair treatments.

Delivering and phenotyping mouse models for the respiratory community: a report on the Biochemical Society Workshop.

The IMPC (International Mouse Phenotyping Consortium) was launched recently, and its aim is to develop and phenotype mouse knockouts of 4000 genes over the next 5 years and, ultimately, of all 20000 or so genes in the mouse genome. As part of the IMPC, the MRC (Medical Research Council) also launched a call for MRC mouse networks, where groups of U.K.-based researchers could form a consortium based around a particular area of research. Members of the respiratory research community formed the RDDRC (Respiratory Development and Disease Research Consortium) to consolidate and develop respiratory phenotyping methods suitable for high-throughput screening. This paper, arising from a Biochemical Society workshop held in London in 2012, highlights the purposes of the RDDRC and the needs of the respiratory research community.

A method for TAT-Cre recombinase-mediated floxed allele modification in ex vivo tissue slices.

Precision-cut lung slices (PCLS) are used for a variety of applications. However, methods to manipulate genes in PCLS are currently limited. We developed a new method, TAT-Cre recombinase-mediated floxed allele modification in tissue slices (TReATS), to induce highly effective and temporally controlled gene deletion or activation in ex vivo PCLS. Treatment of PCLS from Rosa26-flox-stop-flox-EYFP mice with cell-permeant TAT-Cre recombinase induced ubiquitous EYFP protein expression, indicating successful Cre-mediated excision of the upstream loxP-flanked stop sequence. Quantitative real-time PCR confirmed induction of EYFP. We successfully replicated the TReATS method in PCLS from Vangl2flox/flox mice, leading to the deletion of loxP-flanked exon 4 of the Vangl2 gene. Cre-treated Vangl2flox/flox PCLS exhibited cytoskeletal abnormalities, a known phenotype caused by VANGL2 dysfunction. We report a new method that bypasses conventional Cre-Lox breeding, allowing rapid and highly effective gene manipulation in ex vivo tissue models.

Volume targeted versus pressure support non-invasive ventilation in patients with super obesity and chronic respiratory failure: a randomised controlled trial.

INTRODUCTION: Automatic titration modes of non-invasive ventilation, including average volume assured pressure support (AVAPS), are hybrid technologies that target a set volume by automated adjustment of pressure support (PS). These automated modes could offer potential advantages over fixed level PS, in particular, in patients who are super obese. METHODS: Consecutive patients with obesity hypoventilation syndrome were enrolled in a two-centre prospective single-blind randomised controlled trial of AVAPS versus fixed-level PS using a strict protocolised setup. MEASUREMENTS: The primary outcome was change in daytime arterial PCO(2) (PaCO(2)) at 3 months. Body composition, physical activity (7-day actigraphy) and health-related quality of life (severe respiratory insufficiency questionnaire, SRI) were secondary outcome measures. RESULTS: 50 patients (body mass index 50±7 kg/m(2); 55±11 years; 53% men) were enrolled with a mean PaCO(2) of 6.9±0.8 kPa and SRI of 53±17. 46 patients (23 AVAPS and 23 PS) completed the trial. At 3 months, improvements in PaCO(2) were observed in both groups (AVAPS 0.6 kPa, 95% CI 0.2 to 1.1, p<0.01 vs PS 0.6 kPa, 95% CI 0.1 to 1.1, p=0.02) but no between-group difference (-0.1 kPa, 95% CI -0.7 to 0.6, p=0.87). SRI also improved in both groups (AVAPS 11, 95% CI 6 to 17, p<0.001 vs PS 7, 95% CI 1 to 12, p=0.02; between groups 5, 95% CI -3 to 12, p=0.21). Secondary analysis of both groups combined showed improvements in daytime physical activity that correlated with reduction in fat mass (r=0.48; p=0.01). CONCLUSION: The study demonstrated no differences between automated AVAPS mode and fixed-level PS mode using a strict protocolised setup in patients who were super obese. The data suggest that the management of sleep-disordered breathing may enhance daytime activity and promote weight loss in super-obese patients.

Biomarkers of acute lung injury: worth their salt?

The validation of biomarkers has become a key goal of translational biomedical research. The purpose of this article is to discuss the role of biomarkers in the management of acute lung injury (ALI) and related research. Biomarkers should be sensitive and specific indicators of clinically important processes and should change in a relevant timeframe to affect recruitment to trials or clinical management. We do not believe that they necessarily need to reflect pathogenic processes. We critically examined current strategies used to identify biomarkers and which, owing to expedience, have been dominated by reanalysis of blood derived markers from large multicenter Phase 3 studies. Combining new and existing validated biomarkers with physiological and other data may add predictive power and facilitate the development of important aids to research and therapy.